No abstract

Smart fluid manipulation with automatically controlled paper valves will enable automated and multi-step immunoassays on paper-based microfluidic devices. In this work, we present an integrated paper-based microfluidic platform with shape-memory polymer (SMP)-actuated fluid valves capable of automated colorimetric enzyme-linked immunosorbent assays (ELISAs). A single-layer microfluidic paper-based analytical device (μPAD) was designed to store all the reagents on the chip, and sequentially transfer reagents to a paper test zone following a specific ELISA protocol through automatic fluidic flow control by the multiple SMP-actuated valves. The actuation of a paper valve was based on the thermally responsive, duel-state shape transformation of a SMP sheet attached to the root of a paper cantilever beam for driving a hydrophilic paper bridge to connect and disconnect two paper channels. A portable colorimetric reader was developed to control the on-chip valve operations, quantify the colorimetric signal output, display the assay result, and wirelessly transmit the data to a smart phone for the application of telemedicine. Reliable operations of the paper valve and the entire μPAD were demonstrated with success rates of 97% and 93%, respectively. A detection mechanism for valve malfunction was designed and confirmed effective to identify any mal-operation of individual valves, thus rendering our platform reliable in real assays. For device calibration, we conducted direct ELISAs of rabbit IgG in phosphate-buffered saline (PBS), and achieved a low limit of detection (LOD) of 27 pM (comparable to that of standard and paper-based ELISAs). In order to demonstrate the clinical application of our multi-step immunoassay platform, we also conducted sandwich ELISAs to quantify the protein level of an inflammatory cytokine, namely tumor necrosis factor (TNF)-α, in surgically injured laryngeal tissues of rats. The protein levels of TNF-α were shown similar between the conventional and μPAD ELISAs.

The human vocal folds (VFs) undergo complex biomechanical stimulation during phonation. The aim of the present study was to develop and validate a phono-mimetic VF flow perfusion bioreactor, which mimics the mechanical microenvironment of the human VFs in vitro. The bioreactor uses airflow-induced self-oscillations, which have been shown to produce mechanical loading and contact forces that are representative of human phonation. The bioreactor consisted of two synthetic VF replicas within a silicone body. A cell-scaffold mixture (CSM) consisting of human VF fibroblasts, hyaluronic acid, gelatin, and a polyethylene glycol cross-linker was injected into cavities within the replicas. Cell culture medium (CCM) was perfused through the scaffold by using a customized secondary flow loop. After the injection, the bioreactor was operated with no stimulation over a 3-day period to allow for cell adaptation. Phonation was subsequently induced by using a variable speed centrifugal blower for 2 h each day over a period of 4 days. A similar bioreactor without biomechanical stimulation was used as the nonphonatory control. The CSM was harvested from both VF replicas 7 days after the injection. The results confirmed that the phono-mimetic bioreactor supports cell viability and extracellular matrix proteins synthesis, as expected. Many scaffold materials were found to degrade because of challenges from phonation-induced biomechanical stimulation as well as due to biochemical reactions with the CCM. The bioreactor concept enables future investigations of the effects of different phonatory characteristics, that is, voice regimes, on the behavior of the human VF cells. It will also help study the long-term functional outcomes of the VF-specific biomaterials before animal and clinical studies.

Human-iPS survived in injured rat vocal folds. The HA hydrogel with hiPS and EGF ameliorated the fibrotic response. Additional work is necessary to optimize hiPS differentiation and further confirm the safety of hiPS for clinical applications.

Vocal loading tasks are often used to investigate the relationship between voice use and vocal fatigue in laboratory settings. The present study investigated the concept of a novel quantitative dose-based vocal loading task for vocal fatigue evaluation. Ten female subjects participated in the study. Voice use was monitored and quantified using an online vocal distance dose calculator during six consecutive 30-min long sessions. Voice quality was evaluated subjectively using the CAPE-V and SAVRa before, between, and after each vocal loading task session. Fatigue-indicative symptoms, such as cough, swallowing, and voice clearance, were recorded. Statistical analysis of the results showed that the overall severity, the roughness, and the strain ratings obtained from CAPE-V obeyed similar trends as the three ratings from the SAVRa. These metrics increased over the first two thirds of the sessions to reach a maximum, and then decreased slightly near the session end. Quantitative metrics obtained from surface neck accelerometer signals were found to obey similar trends. The results consistently showed that an initial adjustment of voice quality was followed by vocal saturation, supporting the effectiveness of the proposed loading task. These tools require specific vocal stimuli. For example, the CAPE-V requires the completion of three defined phonation tasks assessed through perceptual rating. This therefore limits the applicability of these tools in situations where the vocal stimuli are varied or unspecified. Many studies have investigated uncertainties in subjective judgment methodologies for voice quality evaluation. Kreiman and Gerratt investigated the source of listener disagreement in voice quality assessment using unidimensional rating scales, and found that no single metric from natural voice recordings allowed the evaluation of voice quality [6]. Kreiman also found that individual standards of voice quality, scale resolution, and voice attribute magnitude also significantly influenced intra-rater agreement [7]. Objective metrics obtained using various acoustic instruments have been investigated, and attempts have been made to correlate these with perceptual voice quality assessments [8][9][10][11][12].A plethora of temporal, spectral, and cepstral metrics have been proposed to evaluate voice quality [13,14]. Commonly used features or vocal metrics include fundamental frequency ( f 0), loudness, jitter, shimmer, vocal formants, harmonic-to-noise ratio (HNR), spectral tilt (H1-H2, harmonic richness factor), maximum flow declination rate (MFDR), duty ratio, cepstral peak prominence (CPP), Mel-frequency cepstral coefficients (MFCCs), power spectrum ratio, and others [15][16][17][18][19]. Self-reported feelings of decreased vocal functionality have been used as a criterion for vocal fatigue in many previous studies [1,4,[20][21][22]. Standard self-administered questionnaires, such as the SAVRa and the Vocal Fatigue Index (VFI), have been used to identify individuals with vocal fatigue, and to characterize their sy...